Regulated cell death pathways with morphological characteristics of necrosis have recently emerged as important fundamental biological processes. The relevance of these pathways in human diseases, e.g. acute neurological injuries, is beginning to be appreciated. Identification of small organic molecules that can be used as probes to interrogate the roles of key biological targets are crucial for achieving a mechanistic understanding of regulated necrotic cell death pathways and for formulating therapeutic strategies for treating human diseases. Over the past several years we have described a regulated necrotic cell death pathway (termed necroptosis), identified a key molecular target (RIP1 kinase) in the pathway and reported on four distinct series of small molecule inhibitors that target RIP1 kinase. Three of these series inhibit RIP1 kinase activity directly, while one of these series inhibits the pathway indirectly. Recently we have identified a new indirect inhibitor (Nec-10). Preliminary data suggests that the mechanism of action for this indirect inhibitor may be through inhibiting RIP1 ubiquitination, which has been shown to be critical for RIP1 signaling. The primary goal of this proposal is to optimize Nec-10 for potency, to design and synthesize mechanistic probes based on Nec-10 analogs and to utilize these compounds for elucidating the role of RIP1 ubiquitination in regulated necrotic cell death. PUBLIC HEALTH RELEVANCE: Cells are generally thought to die through two processes, called apoptosis (a regulated process) and necrosis (a non-regulated process). Although the necrosis mechanism is prevalent in many neurological diseases, such as stroke and traumatic brain injury, it has not been targeted by therapies due to the belief that it can not be modulated. However, we and others have found that in some cases this is not true and that therapeutics could potentially be developed for the necrosis form of cell death if its molecular mechanism was better understood. We propose to develop small organic compounds that can be used as molecular probes to investigate necrosis so that therapeutic strategies can be devised for the treatment of neurological diseases where this form of cell death is present.